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Medical microbiologists have relied heavily on the use of Koch's approach to cultivate, propagate and establish the involvement of organisms in disease. However, clinical observation is proving that some pathological conditions are associated with microbes that have not been detected by traditional cultural methods. Two recent advances have enabled the characterization of uncultured microorganisms: the introduction of the polymerase chain reaction for gene amplification and the establishment of a phylogenetically correct classification scheme for bacteria. This review seeks to summarize the advances that have been made in the characterization of uncultured microorganisms, including examples of new bacteria and disease associations which have been established. The review also describes how methods originally employed in microbial ecology have been applied to the analysis of complex bacterial communities, such as the oral microflora, and to the microbiology of purulent polymicrobial infections, without the biases of culture. The scope and limitations of these methods in future applications are discussed. We hope to illustrate how molecular biology and microbial ecology are being combined and applied to improve our understanding of human microbial disease, and how this may ultimately require a revision of Koch's postulates.